Dietary ellagic acid supplementation attenuates intestinal damage and oxidative stress by regulating gut microbiota in weanling piglets

Abstract

Intestinal oxidative stress triggers gut microbiota dysbiosis, which is involved in the etiology of post-weaning diarrhea and enteric infections. Ellagic acid (EA) can potentially serve as an antioxidant supplement to facilitate weaning transition by improving intestinal oxidative stress and gut microbiota dysbiosis. Therefore, we aimed to investigate the effects of dietary EA supplementation on the attenuation of intestinal damage, oxidative stress, and dysbiosis of gut microbiota in weanling piglets. A total of 126 piglets were randomly assigned into 3 groups and treated with a basal diet and 2 mL saline orally (Ctrl group), or the basal diet supplemented with 0.1% EA and 2 mL saline orally (EA group), or the basal diet and 2 mL fecal microbiota suspension from the EA group orally (FEA group), respectively, for 14 d. Compared with the Ctrl group, EA group improved growth performance by increasing average daily feed intake and average daily weight gain (P < 0.05) and decreasing fecal scores (P < 0.05). EA group also alleviated intestinal damage by increasing the tight junction protein occludin (P < 0.05), villus height, and villus height-to-crypt depth ratio (P < 0.05), while decreasing intestinal epithelial apoptosis (P < 0.05). Additionally, EA group enhanced the jejunum antioxidant capacity by increasing the total antioxidant capacity (P < 0.01), catalase (P < 0.05), and glutathione/oxidized glutathione (P < 0.05), but decreased the oxidative metabolite malondialdehyde (P < 0.05) compared to the Ctrl group. Compared with the Ctrl group, EA and FEA groups increased alpha diversity (P < 0.05), enriched beneficial bacteria (Ruminococcaceae and Clostridium ramosum), and increased metabolites short-chain fatty acids (P < 0.05). Correspondingly, FEA group gained effects comparable to those of EA group on growth performance, intestinal damage, and intestinal antioxidant capacity. In addition, the relative abundance of bacteria shifted in EA and FEA groups was significantly related to the examined indices (P < 0.05). Overall, dietary EA supplementation could improve growth performance and attenuate intestinal damage and oxidative stress by regulating the gut microbiota in weanling piglets.

Keywords: Ellagic acid; Gut microbiota; Intestinal damage; Oxidative stress; Weanling piglets.

Fig. 1

The effects of dietary ellagic acid (EA) supplementation and fecal microbiota transplantation (FMT) on diarrhea, intestinal damage, and redox imbalance in weanling piglets. (A) Fecal scores on d 7 and 14. (B) Representative bands for Western blot. (C and D) Western blot of tight junction proteins claudin-1 and occludin in jejunum tissue, respectively. (E-I) Antioxidant indices including GSH/GSSG, T-AOC, CAT, MDA, and NO in the jejunum tissue. Ctrl, the control group, where piglets were fed the basal diet; EA, the EA group, where piglets were fed the basal diet supplemented with EA; FEA, the FEA group, where piglets were fed the basal diet and received FMT from EA-treated piglets. GSH/GSSG = glutathione/glutathione (oxidized); T-AOC = total antioxidant capacity; CAT = catalase; MDA = malondialdehyde; NO = nitric oxide. Data presented as mean ± SEM and significance was presented as ∗P < 0.05 and ∗∗P < 0.01 (n = 3).

Fig. 2

The effect of dietary ellagic acid (EA) supplementation and fecal microbiota transplantation (FMT) on intestinal morphology and epithelial apoptosis in weanling piglets. (A) H&E and TUNEL stained jejunum tissue. (B) Villus height. (C) Crypt depth. (D) Villus height-to-crypt depth ratio. (E) AOD of TUNEL staining. Scaler bar: 500 μm (H&E), 100 μm (TUNEL). Ctrl, the control group, where piglets were fed the basal diet; EA, the EA group, where piglets were fed the basal diet supplemented with EA; FEA, the FEA group, where piglets were fed the basal diet and received FMT from EA-treated piglets. AOD = average optical density, H&E = hematoxylin and eosin stain; TUNEL = terminal deoxynucle otidyl transferase dUTP nick end labeling; DAPI = 4′,6-diamidino-2-phenylindole. Data presented as mean ± SEM and significance was presented as ∗P < 0.05 (n = 7).

Fig. 3

The effect of dietary ellagic acid (EA) supplementation and fecal microbiota transplantation (FMT) on the gene expression of inflammatory cytokines and antioxidant factors in weanling piglets. The genes of antioxidant indices in jejunum tissues including the following: (A) HO-1, (B) NQO-1, (C) GCLC, and (D) GCLM. The genes of inflammatory cytokines in jejunum tissues including the following: (E) TNF-α, (F) IL-6, (G) IL-1α, (H) IL-1β, and (I) IL-10. Ctrl, the control group, where piglets were fed the basal diet; EA, the EA group, where piglets were fed the basal diet supplemented with EA; FEA, the FEA group, where piglets were fed the basal diet and received FMT from EA-treated piglets. HO-1 = heme oxygenase −1; NQO-1 = quinone oxidoreductase-1; GCLC = glutamate cysteine ligase catalytic subunit; GCLM = glutamate cysteine ligase regulatory subunit; TNF-α = tumor necrosis factor–α; IL-6 = Interleukin-6; IL-1β = interleukin-1β; IL-1α = interleukin-1a; and IL-10 = interleukin-10. Data presented as mean ± SEM and significance was presented as ∗P < 0.05 and ∗∗P < 0.01 (n = 7).

Fig. 4

The effect of dietary ellagic acid (EA) supplementation and fecal microbiota transplantation (FMT) on the gut microbiota in weanling piglets. (A) Rarefaction curve of species counts. (B) Venn diagram of OTU distribution among groups. (C) The structure shifts (beta diversity) presented by PCoA plot based on Bray–Curtis distances and assessed by PERMANOVA analysis. Ctrl, the control group, where piglets were fed the basal diet; EA, the EA group, where piglets were fed the basal diet supplemented with EA; FEA, the FEA group, where piglets were fed the basal diet and received FMT from EA-treated piglets. OTU = operational taxonomic units; PCoA = principal coordinates analysis. Data presented as mean ± SEM. (n = 7).

Fig. 5

The effect of dietary ellagic acid (EA) supplementation and fecal microbiota transplantation (FMT) on the taxon abundance of gut microbiota in weanling piglets. (A and B) The relative abundance of gut microbiota at levels of phylum and species, respectively. (C-J) The relative abundance of differential bacteria among groups. Ctrl, the control group, where piglets were fed the basal diet; EA, the EA group, where piglets were fed the basal diet supplemented with EA; FEA, the FEA group, where piglets were fed the basal diet and received FMT from EA-treated piglets. Data presented as mean ± SEM and significance was presented as *P < 0.05 and **P < 0.01 (n = 7).

Fig. 6

The effect of dietary ellagic acid (EA) supplementation and fecal microbiota transplantation (FMT) on the function of gut microbiota in weanling piglets. Different functional composition of gut microbiota: (A) Ctrl vs. EA, (B) Ctrl vs. FEA. Ctrl, the control group, where piglets were fed the basal diet; EA, the EA group, where piglets were fed the basal diet supplemented with EA; FEA, the FEA group, where piglets were fed the basal diet and received FMT from EA-treated piglets. Data presented as mean ± SEM (n = 7).

Fig. 7

Correlation analysis among the identified different bacteria and examined indices. Red and blue represent positive and negative correlations, respectively. P = phylum; f = family; g = genus; s = species. Significance was presented as *P < 0.05, **P < 0.01, and ***P < 0.001 (n = 7).

Fig. S1. The effects of dietary different levels of ellagic acid (EA) supplementation on diarrhea of weanling piglets: fecal scores on d 7 and 14 post weaning. Data presented as mean ± SEM and significance was presented as ∗P < 0.05 (n = 5).